Using combined chromatographic methods, three steroids were isolated from the methanol extract of the sponge Clathria vulpina. Their structures were elucidated to be 3beta-hydroxycholest-5-ene-7-one (1), stigmast-4-ene-3,6-dione (2), and stigmast-4-ene-3-one (3) by 1D- and 2D-NMR spectroscopic methods and in comparison with those reported in the literature. All compounds were evaluated for cytotoxic activities on eight human cancer cell lines, HepG-2, KB, LU-1, MCF-7, LNCaP, SW-480, MKN-7, and HL-60. As the results, compound 3 exhibited moderate cytotoxic activity with the IC50 values ranging of 37.12 - 45.19 ug/mL.